Table 3 Eligible PICO/PECO (I/E: Intervention or Exposure) and study types

Eligible populations

Any human, animal (domestic, livestock, aquaculture) or plant susceptible to receive antibiotics as preventive or curative treatment

Any matrix contaminated by ATBR and destined to a process:

Liquid matrices: e.g., wastewater, livestock slurry, waste milk (unsafe for consumption after antibiotic treatment), source of drinking water

Solid matrix: e.g., sewage sludge, livestock manure, any organic waste product

Any natural compartment contaminated by ATBR:

Aquatic environment: e.g., lake, river, coastal environment, sea

Terrestrial environment: e.g., agricultural soils, soils from natural parks

Wildlife

Eligible interventions (primary objective)

Any intervention to reduce the antibiotic use, e.g.:

Ban of antibiotics, particularly as growth promoters in husbandries

Alternative or preventive treatment (herbal medicines, essential oils, probiotics, prebiotics)

Optimization of the antibiotic use (doses and treatment duration)

Vaccination

Better hygiene conditions

Livestock management practices (free-range or organic farms)

Any process on matrices contaminated by ATBR, e.g.:

Primary (e.g., coagulation, flotation), secondary (e.g., activated sludge, membrane, ponds), tertiary treatments (e.g., advanced oxidation processes, nano/ultrafiltration, activated carbon) in wastewater or drinking water treatment plants

Composting

Aerobic or anaerobic digestion

Liming

Drying

Any environmental management option, e.g.:

Phytoremediation

Buffer zone

Frequency of organic waste spreading

Solutions to be identified

OR Eligible exposures (other objective)

To antibiotics as preventive or curative treatments

To pollutants responsible for the selection or co-selection of resistance, e.g.: antibiotics, metals, biocides

To sources of contamination by ATBR, e.g. discharge of the WWTP, organic waste spreading on agricultural soils, various municipal waste in landfills, aquaculture activities, agricultural and farming activities, urbanization and rejection of untreated wastewaters

Eligible comparators

Alone or in combination (e.g., BACI for before/after/control/intervention): Before I/E, Control or Comparator without I/E, comparator with another I/E

Absence of comparator led to exclusion of the article

Eligible outcomes for ATBR in environment

ATBR defined as:

Antibiotic resistant bacteria (ARB) measured through culture-based methods:

 Concentration or absolute abundance, e.g., in colony forming unit (CFU) per volume or mass of sample

 Proportion or relative abundance in % resistant isolates in the measured bacterial population (e.g., total culturable bacteria, total Escherichia coli isolates)

Antibiotic resistance genes (ARG) and mobile genetic elements (MGE) measured through molecular methods:

 Concentration or absolute abundance in gene copies per volume or mass of samples

 Proportion or relative abundance in gene copies per total microbial biomass, e.g., estimated by the 16S sub-unit of ribosomal ribonucleic acid (16S rRNA genes copies)

Concentrations/Absolute abundances were used for risk assessment associated to the presence of ARB, ARG and MGE

Proportions/Relative abundances were used for quantitative comparisons since the I/E can also impact the total microbial biomass and to overcome the difference between matrices

Environmental samples defined as liquid (e.g. wastewater) and solid matrices (e.g. sewage sludge, livestock manure), natural environment samples including water and sediment from aquatic environment, soil and biological samples from wildlife

Eligible study types

 Inclusion: laboratory-scale and field-scale environmental studies; modelling studies only if a dataset was acquired from experiments or observations

 Exclusion: clinical studies, pure simulation studies, reviews and opinion papers even if considered as potential secondary information sources